Optical techniques that measure cellular phenotype and function most often employ labeled antibodies that specifically label proteins of interest. For example, immunohistochemistry employs samples that have been affixed to a slide and labeled with fluorescently or enzymatically labeled antibodies. Immunohistochemistry has been used in many contexts, including the upregulation of proteins and the characterization of cellular phenotypes. Immunofluorescence microscopy measures antigenic structure with fluorescently labeled antibodies. Uses of this technique include studies of surface expression of proteins in normal and leukemic lymphocytes, and surface protein internalization by activated cells. In confocal microscopy, a laser is scanned across cells labeled with fluorescent antibodies. The specimen is brought into focus by an objective lens and laterally scanned under computer control. Cross-sectional areas are computationally compiled into three-dimensional objects and the subcellular location of labeled proteins is then determined. Confocal microscopy has been used to study antigen expression, apoptosis, and cellular activation.
One of the most common technologies used to phenotype cells is flow cytometry. Flow cytometry measures light scattering, impedance, and fluorescent signals to characterize specific cell types and can identify subpopulations of these cells for sorting. Commercially available flow cytometers measure up to eight different fluorescent molecules bound to a cell, each emitting a distinct wavelength of fluorescent light. Flow cytometers also can measure cellular processes such as cell division, apoptosis, necrosis, and differentiation. Recent developments in flow cytometry sort analyzed cells into microchannels and separate these populations according to fluorescence characteristics.
Flow cytometry has a number of limitations. One drawback is that flow cytometry requires pre-labeling of the cells with fluorescent antibodies or other fluorophores and entails complex experimental protocols that may be difficult to implement in many environments (e.g., resource-poor settings). For flow cytometry to be accurate, the cells must be precisely moved through the center of the focal plane of the illuminating light. This requires a sophisticated system of fluidics, and a narrow orifice in the flow cell (usually in the 50 to 100 micron diameter range). Disturbances in the sample stream can alter the information produced by a flow cytometer, making this technology sensitive to sample clogging and to environmental perturbations. Distinguishing the various cell phenotypes in a sample requires excitation at multiple wavelengths and monitoring of the fluorescence signal in multiple spectral bands. This requires a multi-wavelength laser system and multichannel detection, which results in the need for complex compensation and dramatically increases the number of controls. As multiplexing capacity is increased, adding the required spectral channels substantially increase the complexity, cost, size, power and weight of the instrumentation. More fundamentally, the number of specific phenotypes that can be analyzed in such a system is limited by the number of spectral channels available, which is on the order of 10 in current state-of-the-art flow cytometers.